Metal ion-binding studies highlight important differences between flaviviral RNA polymerases.
West Nile virus (WNV) is a member of the Flaviviridae family which includes a number of important human pathogens. The WNV NS5 protein harbors an RNA-dependent RNA polymerase activity which is required both for replication and transcription of the viral genome. To extend our studies on the role of metal ions in the activity of flaviviral polymerases, we have used fluorescence spectroscopy, circular dichroism, and a combination of chemical and thermal denaturation assays to monitor the consequences of metal ion binding to the enzyme. We demonstrate that the binding of magnesium is not critical for the structural stabilization of the enzyme. Moreover, structural studies indicate that the protein does not undergo conformational change upon the binding of magnesium ions. Additional binding assays also indicate that the interaction of magnesium ions with the enzyme does not significantly stimulate the interaction with the RNA or NTP substrates. The inability of cobalt hexamine, an exchange-inert metal complex structurally analogous to magnesium hexahydrate, to support the catalytic activity also allowed us to demonstrate a direct role of magnesium ions in the catalytic activity of the enzyme. Finally, a three-dimensional structural model of the active center of the enzyme was generated which highlighted the importance of two aspartate residues involved in the coordination of two metal ions. Mutational analyses confirmed the importance of these two amino acids for the binding of magnesium ions. Our data provide further insight into the precise role of magnesium ions for the RNA polymerase activity of the protein, and more importantly, highlight key differences between the RNA polymerases of the Flaviviridae family.
Bougie
Isabelle
I
Département de Biochimie, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.
Bisaillon
Martin
M
D001483:Base Sequence; D001665:Binding Sites; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D005416:Flavivirus / Q000201:enzymology*; D007477:Ions / Q000378:metabolism; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D009154:Mutation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism; D014902:West Nile virus / Q000201:enzymology
D002712:Chlorides; D007477:Ions; D008670:Metals; D017361:Viral Nonstructural Proteins; C007936:hexaminecobalt (III) trichloride; D003035:Cobalt; D014508:Urea; D012321:DNA-Directed RNA Polymerases; D008274:Magnesium